7/26/2001 @ 5:00PM

Biotech's Glowing Breakthrough

These mice are glowing because scientists inserted a gene found in certain bioluminescent jellyfish into their DNA. That gene is a recipe for a protein that glows green when hit by blue or ultraviolet light. The protein is present throughout their bodies. As a result, their skin, eyes and organs give off an eerie light. Only their fur does not glow.

Created by
Tony
Perry
Tony Perry
and
Teru
Wakayama
Teru Wakayama
at
Advanced Cell Technology
in Worcester, Mass., these mice draw attention to how powerful genetic engineering has become. They also underscore the importance of green fluorescent protein, or GFP. The glowing protein is now a widely used biological highlighter that helps scientists find and study genes more quickly. But few noticed when
Osamu
Shimomura
Osamu Shimomura
, then a scientist at Princeton, discovered GFP 40 years ago.

Osamu
Shimomura
Osamu Shimomura
first noticed green fluorescent protein (GFP) in 1962. At first, it was a mere footnote in a scientific paper about a small, bioluminescent jellyfish called Aequoria Victoria. The study of that jellyfish’s glow became Shimomura’s life’s work.

For 20 years starting in 1967, Shimomura made a summer pilgrimage to Friday Harbor in Washington state. With his wife, son and daughter he might gather more than 3,000 jellyfish per day. Over several months, that could add up to 50,000 jellyfish weighing a total of two and a half tons.

From that massive payload of jellyfish, it would be possible to purify perhaps a few hundred milligrams of the glowing proteins for study. A single jellyfish does not need much light-emitting protein to make its lens-shaped body glow.

The average Aequoria Victoria is three to four inches wide and shaped like an umbrella, with 100 light-producing organs the size of poppy seeds spaced on its outer rim. Inside each organ, two chemical reactions produce the green glow.

A protein called aequorin produces the light, through a reaction that involves calcium ions. But this light is blue. Green fluorescent protein absorbs this blue and re-emits it as a green glow. For years, Aequorin received most of the attention. Seven years after GFP was first identified, a team of Harvard researchers “discovered” it, never having heard of it before.

Aequorin proved useful, particularly as a tool for studying nerves, which use the calcium ions it reacts with. GFP would eventually become a vital tool that molecular biologists would use to earmark genes they want to study. But first, the gene that creates the GFP protein needed to be found. That would take decades.

William
Ward
William Ward
(right) met
Douglas
Prasher
Douglas Prasher
on a jellyfish-hunting expedition in the 1980s. Ward, a professor at Rutgers University, had spent a decade becoming one of the world’s experts on the green fluorescent protein found in the Aequoria jellyfish. Prasher wanted to find the gene that makes GFP.

Prasher, a researcher at the Woods Hole Oceanographic Institute, already knew Aequoria well. He cloned the jellyfish’s other glowing protein, Aequorin, while doing his graduate work at the University of Georgia. But cloning the GFP gene would prove difficult to fund and to finish.

After scrounging for funding, Prasher landed a three-year grant from the American Cancer Society. He used up all three years trying to find a genetic sequence that matched the protein–a task that could be done quickly today. When he finished in 1992, he didn’t have enough funding left to put the gene in bacteria–a necessary test if he was to be sure he had the right DNA sequence.

Martin
Chalfie
Martin Chalfie
at Columbia University and
Roger
Tsien
Roger Tsien
at University of California in San Diego wound up trying the protein in other organisms, using Prasher’s sequences. Chalfie is generally credited with popularizing the protein. Prasher did not receive tenure at WHOI and moved on to a completely different field.

He is now a population geneticist with the United States Department of Agriculture in Wood’s Hole, Massachusetts.

In the early 1990s, a Columbia professor named
Martin
Chalfie
Martin Chalfie
heard that another researcher, Douglas Prasher, was trying to locate the gene for a green fluorescent protein (GFP) found in jellyfish. Excited, Chalfie called Prasher and asked for a copy of the gene.

But when Prasher finally found the gene after years of research, Chalfie was away on sabbatical at the University of Utah. “At the time, he was never at the phone,” Prasher says. “He had a girlfriend out there.”

Prasher went ahead and published his description of the GFP gene; Chalfie found that scientific paper while working with a graduate student, and the two researchers finally made contact. Prasher sent Chalfie a copy of the sequenced gene.

Many doubted the GFP gene would produce the glowing protein on its own. But when Chalfie put it in bacteria and shined a blue light on them, they glowed. Chalfie’s 1994 paper on the gene popularized it as a genetic marker. Scientists could link GFP with another gene; were this piece of DNA present in a cell, it would shine.

As for Chalfie’s girlfriend, a noted fruit fly researcher name Tulle Hazelrigg: The two married, and both are professors at Columbia. Hazelrigg made her own large contribution to GFP research: She was among the first to attach GFP to other proteins, allowing scientists to watch where individual proteins go within a cell.

Martin Chalfie’s creation of bacteria that glowed with green fluorescent protein made molecular biology explode with new color. Scientists could attach the GFP gene to other genes. Instead of running complicated tests to see if they had managed to insert a gene into an organism, scientists could just shine a blue light and watch for the glow.

“It’s like having a spell check that underlines words if you’ve made a mistake,” says Rutgers Professor Bill Ward, who was shocked that Chalfie’s bacteria shone at all. “The rest of us knew it wouldn’t work,” Ward says.

Other bioluminescent proteins don’t light up unless certain enzymes are present. But GFP is a concrete wall of a molecule–it curves around itself such that there is no place for an enzyme to bind. “It’s like someone’s feet in Jersey gangster movies where you’re given concrete overshoes,” says GFP researcher Roger Tsien.

Another surprise: Many genes produce half-baked proteins that need to interact with other proteins, made by other genes, to function. But this is not true for GFP–it doesn’t need any help at all.

The green fluorescent protein was originally used to discover whether genes were present at all. Then
Tulle
Hazelrigg
Tulle Hazelrigg
, a professor at Columbia, modified genes in fruit flies so that the proteins they make have GFP glued to them. The result is rather like tying a flashlight to your dog’s head. Even in total darkness, you can see where he is.

Using such fusion proteins, a scientist can follow exactly where a protein moves in a cell, or in an animal’s body. In this case, Hazelrigg was studying a protein involved in the production of sperm and egg. The bright spots on this male larva are the fly’s testes. The rest of the larvae is green because of bioluminescence in its gut; the GFP is expressed mostly in the reproductive organs.

Creating transgenic animals that contain the GFP gene has become increasingly important. In mice, for instance, GFP has enabled adult stem-cell research. Stem cells taken from one mouse and put in another can be identified by their green glow.

Scientists who want to insert green fluorescent protein into cells are no longer restricted to green. The protein now comes in yellow and blue varieties. Generally, the GFP seen in the lab is not the same stuff found in jellyfish.

Roger
Tsien
Roger Tsien
, a professor at University of California in San Diego, mutated and otherwise altered the GFP gene to produce various colors. He also managed to make it brighter. The GFP found in the Aequoria jellyfish produces some of its light when hit by ultraviolet light, some when hit by various shades of blue. Tsien’s version of the protein produces all of its light when hit by a single color.

GFP is a valuable tool, and Tsien’s tinkering made it more valuable. The intellectual property waters surrounding all GFP work are muddy, but Tsien’s patents were strong enough to serve as one of the foundations for a biotech called
Aurora Biosciences
. Aurora was recently bought by
Vertex Pharmaceuticals
for nearly $600 million in stock.